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This study was conducted to test the hypothesis that between year variability in reproductive response to melatonin treatment of young Border Leicester (BL) rams in spring was related to sexual maturity at the time of treatment. Two variables of sexual maturity (mating behaviour and semen quality) were examined. In Experiment 1, mating activity of melatonin-treated and untreated rams was examined at two sites before a flock mating at one site. In Experiment 2, testosterone concentrations, semen quality and variables of in vitro fertilising capacity were examined. In Experiment 1, melatonin treatment did not alter sexual behaviour (latency to nose, total nosings, courtings including Flehmen expression, attempted mounts, mounts, ejaculations) with the exception of a lesser (P < 0.05) Flehmen expression at Site 1 whereas values for most variables were greater (P < 0.05) at Site 2. Treatment resulted in a greater pregnancy rate (89 % and 82 %, respectively; P < 0.05) and advanced distribution of pregnancies (P < 0.001) at Site 1. Testicular size and testosterone concentrations were also greater (P < 0.05) as a result of melatonin treatment in spring whilst the reverse occurred in autumn. In Experiment 2, sperm motility and in vitro fertilising capacity were greater (P < 0.05) and acrosome damage less (P < 0.05) as a result of melatonin treatment. In conclusion, variability in age at sexual maturity of young BL rams at spring mating can be reduced with melatonin treatment and is associated with differences in sexual behaviour, testicular growth, testosterone concentrations, and sperm quality.

Floor space is an important determinant of aggression and stress in group-housed sows, and the aim of the present experiment was to comprehensively examine the effects of floor space in the range of 1.45 to 2.90 m/sow from mixing until 27 d after insemination on aggression, stress, and reproduction of group-housed sows. A previous experiment on the effects of floor space indicated spatial variability across and along the research facility in both sow aggression and stress. To minimize this spatial variability within the research facility, similar-sized pens but with varying groups sizes (10-20) in 4 separate blocks of 3 contiguous pens within each of 9 time replicates (180 sows/replicate) were used to examine 6 space allowances (1.45-2.9 m/sow). Space treatments were appropriately randomized to pens. Although it may be argued that space allowance is confounded with group size in this design, there was no evidence in our previous experiment of group size effects, for pens of 10 to 80 sows, or appreciable interactions between space and group size on aggression, stress, and reproduction. In the present experiment, sows were introduced to treatments within 4 d of insemination and were floor fed 4 times per day (2.5 kg/sow per d). On both Days 2 and 26 after mixing, aggressive behavior (bites and knocks) at feeding and plasma cortisol concentrations were measured. Restricted maximum likelihood mixed model analyses were used to examine the treatment effect after accounting for replicate and random spatial location effects within replicate. There was a consistent linear effect of floor space allowance on aggression at feeding at Day 2 ( < 0.0001) and plasma cortisol concentrations at Day 2 ( = 0.0003), with aggression and stress declining with increasing space. However, there were no effects of space allowance on aggression and stress at Day 26 ( = 0.14 and = 0.79, respectively). These results show that increased floor space in the immediate post-mixing period reduces aggression and stress and that sows may adapt to reduced floor space over time. A strategy of staged-gestation penning, with more space immediately after mixing and less space later in gestation, may address both animal welfare and economic considerations, but this clearly requires further examination.

The roles of testicular hormones in the negative feedback regulation of the secretion and actions of GnRH in male domestic ruminants are reviewed, concentrating mainly on research conducted with rams. Testicular steroids have major feedback actions directly at the hypothalamus to inhibit the secretion of GnRH, although it is apparent that, under certain circumstances, the steroids also have actions directly at the pituitary gland. Further research is necessary to delineate these actions and to determine the contribution of testosterone and its primary metabolites, dihydrotestosterone and oestradiol, to negative feedback on the hypothalamo-pituitary unit. Since GnRH neurones do not possess receptors for steroids, testicular steroids must evoke other neuronal pathways to influence GnRH producing neurones. While the opioids may be important in this regard, it is necessary to determine which other neuronal pathways may also be involved to understand fully the mechanism of action of testicular steroids. It appears that the feedback regulation of the secretion of LH can be accounted for by testicular steroids, whereas the secretion of FSH is influenced by inhibin and steroids, and possibly the recently isolated proteins follistatin and activin. The actions of inhibin to suppress the secretion of FSH occur at the pituitary gland and not on the synthesis or secretion of GnRH. There is a complex interaction between testosterone and inhibin in the control of FSH secretion that results in synergistic effects during the non-breeding season but not during the breeding season. Activin has been shown to have FSH-stimulating properties and follistatin has been shown to have FSH-inhibiting properties, but it is unknown if these proteins play a physiological role in the feedback regulation of FSH in male domestic ruminants.

There is increasing interest in using nonblood measures of glucocorticoids to assess the physiological response to chronic stress conditions. In sheep, cortisol has been measured in various matrices including saliva, feces, and wool, but comprehensive studies of the relationship between plasma concentrations of cortisol and concentrations in these nonblood matrices are lacking. Therefore, we tested the hypothesis that administration of cortisol to sheep would result in elevated concentrations of cortisol in blood, saliva, feces, and wool. Merino ewes were administered with saline or 2 mg/kg BW/d hydrocortisone acetate (HCA) by intramuscular (i.m.) injection for 28 d. This treatment was imposed to mimic circulating cortisol concentrations experienced during periods of chronic stress. Cortisol and cortisone were directly measured in plasma, saliva, and wool before, during, and after treatment with saline or HCA. A 14-d pre-treatment and a 14-d post-treatment period were used to measure time taken for glucocorticoid concentrations in each of the matrices to return to baseline levels. Cortisol was also measured in feces before, during, and after treatment. Wool growth was also measured. Before treatment, there was no difference in the concentration of cortisol or cortisone in plasma, saliva, feces, or wool in animals treated with saline or HCA. In contrast, treatment with HCA increased (P < 0.05) concentrations of both cortisol and cortisone in plasma, saliva, and wool and of cortisol in feces. In plasma, cortisol concentrations were higher than cortisone (P < 0.05), whereas saliva cortisol and cortisone concentrations did not differ significantly. In wool, the concentration of cortisone was about 19-fold higher than that of cortisol during treatment and post-treatment periods. Treatment with HCA inhibited wool growth. These results demonstrate that an increase in glucocorticoids in the blood of sheep is reflected in increases in saliva (after 7 d of treatment), feces (21 d), and wool (14 d). Therefore, measures of glucocorticoids in these matrices may provide a measure of activation of the adrenal glands over time in sheep, thereby providing a retrospective indicator of chronic stress. With respect to wool, it appears that cortisol is predominantly metabolized to cortisone in the skin or wool follicle and is stored as cortisone. Therefore wool cortisone may also provide an important measure in quantifiying chronic stress in sheep.

Activation of the hypothalamo-pituitary-adrenal (HPA) axis by psychosocial stress is attenuated during lactation. We tested the hypothesis that lactating ewes will have attenuated HPA axis responses to isolation and restraint but will have greater responses to predator stress in the form of barking dogs. We imposed two 4 h stressors: psychosocial stress (isolation and restraint of ewes) and predator stress (barking dogs). Blood was collected intravenous every 10 min from nonlactating ewes (n = 6), lactating ewes with lambs present but not able to be suckled (n = 6), and lactating ewes with lambs present and able to be suckled (n = 6). Plasma cortisol and oxytocin were measured. For nonlactating ewes, cortisol increased (P < 0.01) in response to both stressors, and these increases were greater (P < 0.01) than that in the lactating animals. For lactating ewes with lambs present but unable to be suckled, cortisol increased (P < 0.05) in response to both stressors with a greater response to barking dogs (P < 0.05). For lactating ewes with lambs present and able to be suckled, cortisol increased (P < 0.01) in response to barking dogs only. Plasma oxytocin was greater (P < 0.01) in lactating ewes than in nonlactating ewes and did not change in response to the stressors. In conclusion, lactating ewes are likely to have a greater HPA axis response to a stressor that may be perceived to threaten the welfare of themselves and/or their offspring. The role of oxytocin in attenuation of the HPA axis to stress in sheep is unclear from the current research and requires further investigation., Highlights • The response of the hypothalamo-pituitary-adrenal axis to stress is attenuated during lactation in sheep. • The extent of this attenuation is influenced by the opportunity to suckle lambs and the type of stressor. • Stressors that pose an immediate threat to the offspring evoke greater stress responses.

This study directly compared the dynamics of change in plasma corticosterone concentration with the dynamics of change in tissue corticosterone concentration in laying hens. In concert, we measured the rate of gluconeogenesis, glycogenesis, and glycolysis in the liver, kidney, skeletal muscle, and heart. We evaluated these changes acutely, over 3 h in response to an adrenocorticotropic hormone (ACTH) injection, and chronically, over 24 h in response to food and water deprivation. In response to ACTH injection, there was a significant (P < 0.05) increase in plasma corticosterone concentration and a parallel significant increase in corticosterone concentration in the skeletal muscle, kidney, and heart. However, the change in corticosterone concentration in the liver did not parallel the plasma, at times it was greater than the plasma, and there was a second significant increase (P < 0.05) in corticosterone concentration in the liver after 180 min. Under these conditions, the rate of gluconeogenesis in the liver decreased and the rate of glycogenesis increased. In contrast, after 12 h and 24 h of food and water deprivation plasma corticosterone concentration was increased, and this was paralleled by increased corticosterone concentration in the liver, an increase in the rate of gluconeogenesis and a decrease in the rate of glycogenesis. After ACTH injection, glucose concentration in the liver was not significantly depleted but after 12 h or 24 h of food and water deprivation it was significantly depleted (P < 0.05). Plasma corticosterone concentration provided different insight into the effect of the stressor on hen physiology under acute and chronic conditions. Our data suggest that extending our evaluation of stress to the site of corticosterone action, that is, the target tissue, may enhance our ability to evaluate stress and the welfare of laying hens.

We tested the hypothesis that overweight/obese men aged 50–70 years will have a greater salivary cortisol, salivary alpha amylase and heart rate (HR) responses to psychological stress compared with age matched lean men. Lean (BMI=20–25 kg/m2; n=19) and overweight/obese (BMI=27–35 kg/m2; n=17) men (50–70 years) were subjected to a well-characterised psychological stress (Trier Social Stress Test, TSST) at 1500 h. Concentrations of cortisol and alpha amylase were measured in saliva samples collected every 7–15 min from 1400 to 1700 h. HR was recorded using electrocardiogram. Body weight, BMI, percentage body fat, resting systolic and diastolic blood pressure and mean arterial pressure were significantly higher (PP=0.187, P=0.288, P=0.550, respectively). There were no significant differences between the groups for pretreatment values, peak height, difference between pretreatment values and peak height (reactivity) or area under the curve for salivary cortisol, salivary alpha amylase or HR (P>0.05 for all). The results showed that, for men with a moderate level of overweight/obesity who were otherwise healthy, the response of salivary cortisol, salivary alpha amylase and HR to acute psychological stress was not impaired.

In several species, females masculinised by abnormal androgen exposure in utero have poor reproductive performance and gilts born into litters with a male bias are likely exposed to greater androgen concentrations prenatally than gilts born into female-biased litters. At 24 h of age, piglet plasma testosterone concentrations in gilts from male-biased litters (>60% male; n = 22) or female-biased litters (>60% female; n = 27) were not different. At 18 wks of age, all gilts received an injection of 400 IU equine chorionic gonadotrophin plus 200 IU human chorionic gonadotrophin to stimulate oestrus. Two weeks after the injection gilts were slaughtered and ovaries collected for determination of numbers of corpora lutea (CL). Compared to gilts from female-biased litters, gilts from male-biased litters were more likely to ovulate (86.0% vs 59.5%, P = 0.047) and had more CL (13.1 ± 1.5 vs 7.2 ± 1.7, P = 0.015). The present data indicate an effect of birth litter sex-bias on pre-pubertal physiological development, possibly involving organisational effects at the ovarian cellular level impacting on future ovarian function. Potential impacts on subsequent fertility remain to be determined.

Glucocorticoids (corticosterone in birds and rodents and cortisol in all other mammals) are glucoregulatory hormones that are synthesized in response to a range of stimuli including stress and are regularly measured in the assessment of animal welfare. Glucocorticoids have many normal or non-stress-related functions, and glucocorticoid synthesis can increase in response to pleasure, excitement, and arousal as well as fear, anxiety, and pain. Often, when assessing animal welfare, little consideration is given to normal non-stress-related glucocorticoid functions or the complex mechanisms that regulate the effects of glucocorticoids on physiology. In addition, it is rarely acknowledged that increased glucocorticoid synthesis can indicate positive welfare states or that a stress response can increase fitness and improve the welfare of an animal. In this paper, we review how and when glucocorticoid synthesis increases, the actions mediated through type I and type II glucocorticoid receptors, the importance of corticosteroid-binding globulin, the role of 11 β-hydroxysteroid dehydrogenase, and the key aspects of neurophysiology relevant to activating the hypothalamo-pituitary-adrenal axis. This is discussed in the context of animal welfare assessment, particularly under the biological functioning and affective states frameworks. We contend that extending the assessment of animal welfare to key brain regions afferent to the hypothalamus and incorporating the aspects of glucocorticoid physiology that affect change in target tissue will advance animal welfare science and inspire more comprehensive assessment of the welfare of animals.

Neuroadrenergic function in type 2 diabetic (T2D) patients without neuropathy is poorly characterized. We therefore compared sympathetic nervous system activity at rest and during an oral glucose tolerance test in obese metabolic syndrome (MetS) subjects classified as glucose intolerant (impaired glucose tolerance [IGT]; n = 17) or treatment-naive T2D (n = 17). Untreated subjects, matched for age (mean 59 ± 1 year), sex, BMI (32.4 ± 0.6 kg/m2), and family history of diabetes were studied. We measured resting muscle sympathetic nerve activity (MSNA) by microneurography, whole-body norepinephrine kinetics by isotope dilution, insulin sensitivity by euglycemic-hyperinsulinemic clamp (steady-state glucose utilization adjusted for fat-free mass and steady-state insulin concentration [M/I]), and MetS components. T2D subjects had higher resting MSNA burst incidence (67 ± 4 versus 55 ± 3 bursts per 100 heartbeats; P = 0.05) and arterial norepinephrine levels (264 ± 33 versus 167 ± 16 pg/mL; P = 0.02), lower plasma norepinephrine clearance (by 17%; P = 0.03), and reduced neuronal reuptake compared with IGT subjects (by 46%; P = 0.04). Moreover, norepinephrine spillover responses to glucose ingestion were blunted in T2D subjects. The M/I value independently predicted whole-body norepinephrine spillover (r = −0.47; P = 0.008), whereas fasting insulin level related to neuronal norepinephrine reuptake (r = −0.35, P = 0.047). These findings demonstrate that progression to T2D is associated with increased central sympathetic drive, blunted sympathetic responsiveness, and altered norepinephrine disposition.

This review focuses on the importance of cortisol in mediating the inhibitory effects of psychosocial stress on reproduction in females. In particular, we have summarized our research in sheep where we have systematically established whether cortisol is both sufficient and necessary to suppress reproductive hormone secretion and inhibit sexual behaviour. Our findings are put into context with previous work and are used to develop important concepts as well as to identify productive further lines of investigation. It is clear that cortisol is necessary to inhibit some, but not all, aspects of reproduction in female sheep. These actions vary with reproductive state, and there are important interactions with gonadal steroids. The impact of cortisol on the tonic secretion of gonadotrophin-releasing hormone and luteinizing hormone has been investigated extensively, but less is known about the surge secretion of these hormones and their effects on sexual behaviour. Furthermore, there are separate effects of cortisol in the brain (hypothalamus) and at the anterior pituitary, illustrating that there are different mechanisms of action. Thus, although cortisol is important in mediating some of the effects of stress on reproduction, we need to look beyond cortisol and investigate some of the other mechanisms and mediators that relay the effects of stress on reproduction. In this regard, we propose that a group of neurons in the hypothalamus that co-synthesize kisspeptin, neurokinin B and dynorphin, termed KNDy cells, play important roles in mediating the effects of cortisol on reproduction. This hypothesis needs to be rigorously tested.

Aim: Insulin resistance and visceral adiposity are predisposing factors for fatty liver disease. The main objectives of this study were (i) to compare the effects of caloric restriction (CR) alone or together with moderate-intensity aerobic exercise training (CR+EX) on liver enzymes, a surrogate marker of liver injury, in obese metabolic syndrome (MetS) subjects and (ii) to identify anthropometric, metabolic, cardiovascular and dietary predictors of changes in liver enzymes. Methods: Sedentary men and women (n = 63), aged 55 ± 6 (s.d.) years with body mass index 32.7 ± 4.1 kg/m and confirmed MetS, were randomized to 12-week CR, CR+EX or no treatment (Control). Results: Weight loss averaged 7.6% in the CR and 9.1% in the CR+EX group (time effect, p < 0.001; group effect, p = 0.11); insulin sensitivity improved by 49 and 45%, respectively (both p < 0.001). Fitness (maximal oxygen consumption) increased by 19% in the CR+EX group only (p < 0.001). Alanine aminotransferase (ALT) levels decreased by 20% in the CR and 24% in the CR+EX group (time effect, both p < 0.001; group effect, p = 0.68); corresponding values for γ-glutamyltransferase (GGT) were -28 and -33%, respectively (time effect, both p < 0.001; group effect, p = 0.28). Reduction in abdominal fat mass (measured by DXA from L1 to L4) independently predicted ΔALT (r = 0.42, p = 0.005) and ΔGGT (r = 0.55, p < 0.001), whereas change in dietary saturated fat intake was independently associated with ΔALT (r = 0.35, p = 0.03). Conclusions: Reductions in central adiposity and saturated fat intake are key drivers of improvement in liver enzymes during lifestyle interventions. Exercise training did not confer significant incremental benefits in this study.

It is well known that stress inhibits normal reproductive function, including gonadotrophin secretion; however, the mechanisms and mediators involved are largely unknown. Stress impairs the secretion of luteinising hormone (LH), and it has been suggested that the RF-amide gonadotrophin-inhibitory hormone (GnIH), known as RF-amide related peptide-3 (RFRP-3) in mammalian species, may mediate this inhibitory effect of stress. If this is the case, the GnIH/RFRP system would likely be up-regulated during stress. We tested this hypothesis in ovariectomised ewes using a psychosocial stressor: isolation/restraint. Ewes were randomly allocated to control or stress (n=5 per group). Isolation/restraint stress was imposed for 90min after control sampling for 4h, whereas control ewes were sampled continuously for 5.5h. All ewes were then euthanased and brains were collected. As expected, plasma concentrations of cortisol were increased significantly (P

Five experiments were conducted to test the hypothesis that psychosocial stress interferes with the estrous cycle of sheep. In experiment 1, ewes were repeatedly isolated during the follicular phase. Timing, amplitude, and duration of the preovulatory luteinizing hormone (LH) surge were not affected. In experiment 2, follicular-phase ewes were subjected twice to a “layered stress” paradigm consisting of sequential, hourly application of isolation, restraint, blindfold, and predator cues. This reduced the LH pulse amplitude but did not affect the LH surge. In experiment 3, different acute stressors were given sequentially within the follicular phase: food denial plus unfamiliar noises and forced exercise, layered stress, exercise around midnight, and transportation. This, too, did not affect the LH surge. In experiment 4, variable acute psychosocial stress was given every 1–2 days for two entire estrous cycles; this did not disrupt any parameter of the cycle monitored. Lastly, experiment 5 examined whether the psychosocial stress paradigms of experiment 4 would disrupt the cycle and estrous behavior if sheep were metabolically stressed by chronic food restriction. Thirty percent of the food-restricted ewes exhibited deterioration of estrous cycle parameters followed by cessation of cycles and failure to express estrous behavior. However, disruption was not more evident in ewes that also encountered psychosocial stress. Collectively, these findings indicate the estrous cycle of sheep is remarkably resistant to disruption by acute bouts of psychosocial stress applied intermittently during either a single follicular phase or repeatedly over two estrous cycles.

Summary The neonatal nutritional environment is critical for programming the systems regulating body weight, and an inappropriate nutritional environment in early life can lead to overweight or obesity later on. In this study we demonstrate that changes to the neonatal nutritional environment, induced by changing the litter size in which the animal is raised, can alter not only body weight into adulthood, but also anxiety behaviours and stress responsiveness. These effects appear to be somewhat sex-dependent, affecting female rats more strongly than males. Thus, Wistar rats raised in small litters, where they have greater access to their mothers’ milk, weigh more in adulthood than those raised in large litters. Females from these small litters show enhanced exploratory behaviour and reduced anxiety in the elevated plus maze, entering the open arms more often. They also display enhanced central responses to restraint stress including greater activation of the paraventricular nucleus of the hypothalamus and paraventricular nucleus of the thalamus, possibly indicating that the changes we see are related to enhanced arousal. Thus, while neonatal litter size affects long-term body weight regulation, it can also alter long-term activity, anxiety and stress responsiveness, and the degree to which it does so appears to be sex-dependent.

Precise control of pulsatile GnRH and LH release is imperative to ovarian cyclicity but is vulnerable to environmental perturbations, like stress. In sheep, a sustained (29 h) increase in plasma cortisol to a level observed during stress profoundly reduces GnRH pulse frequency in ovariectomized ewes treated with ovarian steroids, whereas shorter infusion (6 h) is ineffective in the absence of ovarian hormones. This study first determined whether the ovarian steroid milieu or duration of exposure is the relevant factor in determining whether cortisol reduces LH pulse frequency. Prolonged (29 h) cortisol infusion did not lower LH pulse frequency in ovariectomized ewes deprived of ovarian hormones, but it did so in ovariectomized ewes treated with estradiol and progesterone to create an artificial estrous cycle, implicating ovarian steroids as the critical factor. Importantly, this effect of cortisol was more pronounced after the simulated preovulatory estradiol rise of the artificial follicular phase. The second experiment examined which component of the ovarian steroid milieu enables cortisol to reduce LH pulse frequency in the artificial follicular phase: prior exposure to progesterone in the luteal phase, low early follicular phase estradiol levels, or the preovulatory estradiol rise. Basal estradiol enabled cortisol to decrease LH pulse frequency, but the response was potentiated by the estradiol rise. These findings lead to the conclusion that ovarian steroids, particularly estradiol, enable cortisol to inhibit LH pulse frequency. Moreover, the results provide new insight into the means by which gonadal steroids, and possibly reproductive status, modulate neuroendocrine responses to stress.

Two experiments were conducted to test the hypothesis that cortisol interferes with the positive feedback action of estradiol that induces the luteinizing hormone (LH) surge. Ovariectomized sheep were treated sequentially with progesterone and estradiol to create artificial estrous cycles. Cortisol or vehicle (saline) was infused from 2 h before the estradiol stimulus through the time of the anticipated LH surge in the artificial follicular phase of two successive cycles. The plasma cortisol increment produced by infusion was ∼1.5 times greater than maximal concentrations seen during infusion of endotoxin, which is a model of immune/inflammatory stress. In experiment 1, half of the ewes received vehicle in the first cycle and cortisol in the second; the others were treated in reverse order. All ewes responded with an LH surge. Cortisol delayed the LH surge and reduced its amplitude, but both effects were observed only in the second cycle. Experiment 2 was modified to provide better control for a cycle effect. Four treatment sequences were tested (cycle 1-cycle 2): vehicle-vehicle, cortisol-cortisol, vehicle-cortisol, cortisol-vehicle. Again, cortisol delayed but did not block the LH surge, and this delay occurred in both cycles. Thus, an elevation in plasma cortisol can interfere with the positive feedback action of estradiol by delaying and attenuating the LH surge.

Our laboratory has developed a paradigm of psychosocial stress (sequential layering of isolation, blindfold, and predator cues) that robustly elevates cortisol secretion and decreases LH pulse amplitude in ovariectomized ewes. This decrease in LH pulse amplitude is due, at least in part, to a reduction in pituitary responsiveness to GnRH, caused by cortisol acting via the type II glucocorticoid receptor (GR). The first experiment of the current study aimed to determine whether this layered psychosocial stress also inhibits pulsatile GnRH release into pituitary portal blood. The stress paradigm significantly reduced GnRH pulse amplitude compared with nonstressed ovariectomized ewes. The second experiment tested if this stress-induced decrease in GnRH pulse amplitude is mediated by cortisol action on the type II GR. Ovariectomized ewes were allocated to three groups: nonstress control, stress, and stress plus the type II GR antagonist RU486. The layered psychosocial stress paradigm decreased GnRH and LH pulse amplitude compared with nonstress controls. Importantly, the stress also lowered GnRH pulse amplitude to a comparable extent in ewes in which cortisol action via the type II GR was antagonized. Therefore, we conclude that psychosocial stress reduces the amplitude of GnRH pulses independent of cortisol action on the type II GR. The present findings, combined with our recent observations, suggest that the mechanisms by which psychosocial stress inhibits reproductive neuroendocrine activity at the hypothalamic and pituitary levels are fundamentally different. A paradigm of psychosocial stress inhibits GnRH pulse amplitude. This effect is not reversed by treatment with RU486, a type II glucocorticoid receptor antagonist.

Stress-like elevations in plasma glucocorticoids suppress gonadotropin secretion and can disrupt ovarian cyclicity. In sheep, cortisol acts at the pituitary to reduce responsiveness to GnRH but does not affect GnRH pulse frequency in the absence of ovarian hormones. However, in ewes during the follicular phase of the estrous cycle, cortisol reduces LH pulse frequency. To test the hypothesis that cortisol reduces GnRH pulse frequency in the presence of ovarian steroids, the effect of cortisol on GnRH secretion was monitored directly in pituitary portal blood of follicular phase sheep in the presence and absence of a cortisol treatment that elevated plasma cortisol to a level observed during stress. An acute (6 h) cortisol increase in the midfollicular phase did not lower GnRH pulse frequency. However, a more prolonged (27 h) increase in cortisol beginning just before the decrease in progesterone reduced GnRH pulse frequency by 45% and delayed the preovulatory LH surge by 10 h. To determine whether the gonadal steroid milieu of the follicular phase enables cortisol to reduce GnRH pulse frequency, GnRH was monitored in ovariectomized ewes treated with estradiol and progesterone to create an artificial follicular phase. A sustained increment in plasma cortisol reduced GnRH pulse frequency by 70% in this artificial follicular phase, in contrast to the lack of an effect in untreated ovariectomized ewes as seen previously. Thus, a sustained stress-like level of cortisol suppresses GnRH pulse frequency in follicular phase ewes, and this appears to be dependent upon the presence of ovarian steroids. A sustained stress-like level of cortisol reduces GnRH pulse frequency in the follicular phase and appears to require the presence of ovarian steroids.

We have shown that cortisol infusion reduced the luteinizing hormone (LH) response to fixed hourly GnRH injections in ovariectomized ewes treated with estradiol during the non-breeding season (pituitary-clamp model). In contrast, cortisol did not affect the response to 2 hourly invariant GnRH injections in hypothalamo-pituitary disconnected ovariectomized ewes during the breeding season. To understand the differing results in these animal models and to determine if cortisol can act directly at the pituitary to suppress responsiveness to GnRH, we investigated the importance of the frequency of GnRH stimulus, the presence of estradiol and stage of the circannual breeding season. In experiment 1, during the non-breeding season, ovariectomized ewes were treated with estradiol, and pulsatile LH secretion was restored with i.v. GnRH injections either hourly or 2 hourly in the presence or absence of exogenous cortisol. Experiments 2 and 3 were conducted in hypothalamo-pituitary disconnected ovariectomized ewes in which GnRH was injected i.v. every 2 h. Experiment 2 was conducted during the non-breeding season and saline or cortisol was infused for 30 h in a cross-over design. Experiment 3 was conducted during the non-breeding and breeding seasons and saline or cortisol was infused for 30 h in the absence and presence of estradiol using a cross-over design. Samples were taken from all animals to measure plasma LH. LH pulse amplitude was reduced by cortisol in the pituitary clamp model with no difference between the hourly and 2-hourly GnRH pulse mode. In the absence of estradiol, there was no effect of cortisol on LH pulse amplitude in GnRH-replaced ovariectomized hypothalamo-pituitary disconnected ewes in either season. The LH pulse amplitude was reduced in both seasons in experiment 3 when cortisol was infused during estradiol treatment. We conclude that the ability of cortisol to reduce LH secretion does not depend upon the frequency of GnRH stimulus and that estradiol enables cortisol to act directly on the pituitary of ovariectomized hypothalamo-pituitary disconnected ewes to suppress the responsiveness to GnRH; this effect occurs in the breeding and non-breeding seasons.

Reproductive activity in sheep is seasonal, being activated by short-day photoperiods and inhibited by long days. During the nonbreeding season, GnRH secretion is reduced by both steroid-independent and steroid-dependent (increased response to estradiol negative feedback) effects of photoperiod. Kisspeptin (also known as metastin) and gonadotropin-inhibitory hormone (GnIH, or RFRP) are two RFamide neuropeptides that appear critical in the regulation of the reproductive neuroendocrine axis. We hypothesized that expression of kisspeptin and/or RFRP underlies the seasonal change in GnRH secretion. We examined kisspeptin and RFRP (protein and mRNA) expression in the brains of ovariectomized (OVX) ewes treated with estradiol (OVX+E) during the nonbreeding and breeding seasons. In OVX+E ewes, greater expression of kisspeptin and Kiss1 mRNA in the arcuate nucleus and lesser expression of RFRP (protein) in the dorsomedial nucleus of the hypothalamus were concurrent with the breeding season. There was also a greater number of kisspeptin terminal contacts onto GnRH neurons and less RFRP-GnRH contacts during the breeding season (compared with the nonbreeding season) in OVX+E ewes. Comparison of OVX and OVX+E ewes in the breeding and nonbreeding season revealed a greater effect of steroid replacement on inhibition of kisspeptin protein and Kiss1 mRNA expression during the nonbreeding season. Overall, we propose that the two RFamide peptides, kisspeptin and RFRP, act in concert, with opposing effects, to regulate the activity of GnRH neurons across the seasons, leading to the annual change in fertility and the cyclical seasonal transition from nonbreeding to breeding season.

Two major characteristics of males that affect the likelihood of achieving copulation are the sexual motivation and mating competency of the male. The behavior of domestic animals, including their sexual behavior, is dependent upon a complex interaction between the organism's internal and external environment. In male pigs, as in other mammalian species, it is clear that testicular steroids are required to maintain sexual behavior and testosterone plays a critical role. Consistent with studies in other species, it is apparent in male pigs that there is a threshold circulating concentration of androgens and/or estrogens required to maintain sexual behavior and that the level of sexual motivation is unrelated to levels of sex steroids providing these are adequate for normal sexual behavior. Key aspects of the external environment that affect the sexual behavior of the male pig include genetic, seasonal, social, sexual and psychological factors.

This study assessed the importance of cortisol in mediating inhibition of pulsatile LH secretion in sheep exposed to a psychosocial stress. First, we developed an acute psychosocial stress model that involves sequential layering of novel stressors over 3–4 h. This layered-stress paradigm robustly activated the hypothalamic-pituitary-adrenal axis and unambiguously inhibited pulsatile LH secretion. We next used this paradigm to test the hypothesis that cortisol, acting via the type II glucocorticoid receptor (GR), mediates stress-induced suppression of pulsatile LH secretion. Our approach was to determine whether an antagonist of the type II GR (RU486) reverses inhibition of LH pulsatility in response to the layered stress. We used two animal models to assess different aspects of LH pulse regulation. With the first model (ovariectomized ewe), LH pulse characteristics could vary as a function of both altered GnRH pulses and pituitary responsiveness to GnRH. In this case, antagonism of the type II GR did not prevent stress-induced inhibition of pulsatile LH secretion. With the second model (pituitary-clamped ovariectomized ewe), pulsatile GnRH input to the pituitary was fixed to enable assessment of stress effects specifically at the pituitary level. In this case, the layered stress inhibited pituitary responsiveness to GnRH and antagonism of the type II GR reversed the effect. Collectively, these findings indicate acute psychosocial stress inhibits pulsatile LH secretion, at least in part, by reducing pituitary responsiveness to GnRH. Cortisol, acting via the type II GR, is an obligatory mediator of this effect. However, under conditions in which GnRH input to the pituitary is not clamped, antagonism of the type II GR does not prevent stress-induced inhibition of LH pulsatility, implicating an additional pathway of suppression that is independent of cortisol acting via this receptor.

This study investigated sex differences in the stress-induced activation of neurons containing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin in the paraventricular nucleus (PVN) of gonadectomized male and female sheep. Groups (n=3) of both sexes were either subjected to 90 min isolation and restraint stress (stress group) or were not stressed. Blood samples were taken every 10 min for 90 min prior to and after stress to monitor cortisol levels in plasma. Brains were harvested after 90 min of stress. Stress caused elevation of plasma cortisol levels to a similar extent in both sexes. Double-labeling immunohistochemistry for Fos and either CRH, AVP or enkephalin was undertaken to quantify the numbers of neurons staining for CRH, AVP and enkephalin that also immunostained for Fos. Stress increased Fos immunostaining in all cell types. There was a greater proportion of CRH than AVP neurons activated in stressed animals. There were no sex differences in the activation of CRH and AVP neurons although females had a greater proportion of enkephalin cells staining for Fos than males in both control and stressed animals. There were no differences between control and stressed animals in the proportion of cells co-staining for CRH and AVP. We conclude that isolation and restraint stress activates neurons producing CRH, AVP and enkephalin in sheep and that CRH may play a greater role than AVP in regulating adrenocorticotrophic hormone secretion in response to this stressor in sheep. Finally, isolation and restraint stress does not influence co-localization of CRH and AVP in sheep.

We investigated the effect of the presence and absence of lambs and suckling by lambs to attenuate activation of the hypothalamo-pituitary-adrenal (HPA) axis to isolation and restraint stress in lactating sheep. In experiment 1, blood samples were collected every 10 min from nonlactating (n = 5) and lactating (n = 5) ewes for 4 h before and during stress. In experiment 2, ewes (n = 6) were allocated to 1) nonlactating, 2) lactating with lambs absent, 3) lactating with lambs present but unable to suckle, and 4) lactating with lambs present and able to suckle. Blood samples were collected over 8 h with no stress (control day) and for 4 h before and 4 h during stress (stress day). In experiment 1, the mean (±sem) cortisol concentrations increased significantly (P < 0.05) in nonlactating ewes during stress but did not change in lactating ewes. In experiment 2, cortisol did not vary on the control day or pretreatment of the stress day but increased (P < 0.05) during stress in all groups except lactating ewes with lambs present and able to suckle. The greatest cortisol response occurred in nonlactating ewes followed by lactating ewes with lambs absent and lactating ewes with lambs present but unable to suckle. During stress, the ACTH concentrations increased (P < 0.05) in nonlactating ewes and lactating ewes with lambs absent but not in lactating ewes with lambs present. We conclude that the activity of the HPA axis during isolation and restraint is reduced in lactating ewes and that the presence of lambs increases this level of attenuation.

It is important to understand factors that may influence responses to stress, as these factors may also influence vulnerability to pathologies that can develop when stress responses are excessive or prolonged. It is clear that, in adults, the sex of an individual can influence the cortisol response to stress in a stressor specific manner. Nevertheless, the stage of development at which these sex differences emerge is unknown. We tested the hypothesis that there are sex differences in the cortisol response to tail docking and ACTH in lambs of 1 and 8 weeks of age. We also established cortisol responses in males when tail docking was imposed alone and in combination with castration at these ages. In experiment 1, 1 and 8 week old male and female lambs were subjected to sham handling, tail docking or, in males, a combination of tail docking and castration. In experiment 2, we administered ACTH (1.0 IU/kg) to male and female lambs at 1 and 8 weeks of age. There were significant cortisol responses to all treatments at both ages. Sex differences in the cortisol responses to tail docking and ACTH developed between 1 and 8 weeks of age, with females having greater responses than males. The data suggest that the mechanism for the sex difference in response to tail docking may involve the adrenal glands. At both ages, in males, the cortisol response to the combined treatment of tail docking and castration was significantly greater than that for tail docking alone.

The paraventricular nucleus (PVN) is integral to regulation of the hypothalamo-pituitary-adrenal (HPA) axis and contains cells producing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin. We used immunohistochemistry to map these peptides and to resolve the extent of co-localization within PVN cells in intact and gonadectomized male and female sheep. Immunoreactive (ir) CRH, AVP and enkephalin cells were mapped in two rams and two ewes at 180 mum intervals throughout the rostro-caudal extent of the PVN. Similar distributions of AVP-ir cells occurred in both sexes whereas CRH-ir and enkephalin-ir cells extended more rostrally in rams. In groups (n=4) of intact and gonadectomized sheep of both sexes, co-localization and distribution of neuropeptides was influenced by sex and gonadectomy. Males had more AVP and CRH cells than females. Intact animals had more AVP cells than gonadectomized animals. There were no differences between groups in the number or percentage of cells that stained for both CRH and AVP or in the number of cells that stained for both CRH and enkephalin. Differences were observed in the percentage of enkephalin cells that contained CRH with males having a greater percentage of co-localized cells than did females. Differences were also observed in the number and percentage of cells that stained for both enkephalin and AVP; the number of cells that stained for both neuropeptides was greater in males than in females and greater in intact animals than in gonadectomized animals. Differences were observed in the percentage of AVP cells that contained enkephalin, and in the percentage of enkephalin cells that contained AVP with males having a greater percentage of co-localized cells than did females. We conclude that sex and gonadal status affect peptide distribution in the PVN of the sheep which may provide an anatomical basis for sex differences in HPA axis responses to stress.

Stress-like elevations in plasma cortisol suppress LH pulse amplitude in ovariectomized ewes by inhibiting pituitary responsiveness to GnRH. Here we sought to identify the receptor mediating this effect. In a preliminary experiment GnRH and LH pulses were monitored in ovariectomized ewes treated with cortisol plus spironolactone, which antagonizes the type I mineralocorticoid receptor (MR), or with cortisol plus RU486, which antagonizes both the type II glucocorticoid receptor (GR) and the progesterone receptor (PR). Cortisol alone reduced LH pulse amplitude, but not pulsatile GnRH secretion, indicating that it reduced pituitary responsiveness to endogenous GnRH. RU486, but not spironolactone, reversed this suppression. We next tested whether RU486 reverses the inhibitory effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, frequency, and duration. Hourly GnRH pulses were delivered to ovariectomized ewes in which endogenous GnRH pulses were blocked by estradiol during seasonal anestrus. Cortisol alone reduced the amplitude of LH pulses driven by the exogenous GnRH pulses. RU486, but not an antagonist of PR (Organon 31710), prevented this suppression. Thus, the efficacy of RU486 in blocking the suppressive effect of cortisol is attributed to antagonism of GR, not PR. Together, these observations imply that the type II GR mediates cortisolinduced suppression of pituitary responsiveness to GnRH.

We aimed to determine the importance of leptin in the regulation of luteinizing hormone (LH) and growth hormone (GH) secretion in ovariectomized (OVX) ewes. Lean and fat sheep were produced by dietary manipulation over 8 months and were then fasted for 32 h. Plasma concentrations of glucose, insulin and leptin were higher in the fat group. Fasting decreased plasma concentrations of glucose and insulin and increased concentrations of nonesterified fatty acids (NEFA) in fat and lean ewes, but leptin concentrations were reduced in the fat group only. Plasma GH concentrations were higher in the lean group and LH concentrations were lower; there was no effect of fasting. These data suggested that long-term changes in plasma leptin concentrations might affect LH and GH secretion, but acute changes with fasting had no effect. OVX ewes of normal body weight were fasted for 72 h with or without intracerebroventricular (i.c.v.) infusion of leptin (4 microg/h), achieving similar metabolic effects to the 32 h fast. The 72-h fast increased LH pulse amplitude, mean GH and cortisol concentrations, but these changes were corrected towards normal by leptin treatment. Thus, leptin could attenuate fasting-induced alterations in the secretion of LH, GH and cortisol. Finally, we food-restricted OVX ewes for 4 months (lean), leading to a 20-kg reduction in body weight. Plasma concentrations of leptin and insulin were decreased, and plasma GH concentrations increased, but there was no effect on plasma concentrations of LH, glucose or NEFA. Icv infusion of leptin did not affect any endocrine or metabolic parameter in these ewes. In summary, maintenance of a lean or fat condition for a prolonged period (8 months) or an extended fasting (72 h) can affect LH and GH secretion, but short-term food restriction (4 months) affected only GH secretion and short-term fasting (32 h) had no effect on either LH or GH secretion. This is in spite of altered plasma leptin concentrations in all circumstances studied. Although leptin treatment can restore plasma concentrations of LH, GH and cortisol towards normal in sheep fasted for 72 h, some other factor(s) must signal to the brain to cause shifts in neuroendocrine function in other conditions where nutritional/metabolic status is altered.

Enkephalin appears to exert an inhibitory action on LH secretion, but whether testosterone regulates enkephalin gene expression is unknown. This study tested the hypothesis that testosterone and/or season modulate preproenkephalin mRNA expression in specific areas of the hypothalamus. Romney Marsh rams were castrated (wethers) either during the breeding season or nonbreeding season and received intramuscular injections of either oil or testosterone propionate (five/group). Blood samples were taken for the assay of plasma LH and testosterone. Preproenkephalin mRNA expression was quantified in hypothalamic sections by in situ hybridization. Mean plasma LH concentrations were reduced and the interpulse interval for LH pulses was greater in testosterone propionate-treated wethers compared with oil-treated wethers, with no change in LH pulse amplitude. Testosterone propionate treatment reduced proenkephalin expression in the diagonal band of Broca, the caudal preoptic area, and the bed nucleus of the stria terminalis. Seasonal differences in proenkephalin expression were observed in the bed nucleus of the stria terminalis, lateral septum, periventricular nucleus, and paraventricular nucleus. No differences were observed between treatments in seven other regions examined. We conclude that testosterone and season regulate proenkephalin mRNA levels in the preoptic area/hypothalamus in the ram in a region-specific manner.

Studies in rats suggest that neuropeptide Y (NPY) plays a stimulatory role in the generation of the preovulatory luteinizing hormone (LH) surge, via the Y1 receptor. We have investigated this issue using the oestradiol benzoate (EB)-treated ovariectomized (OVX) ewe which is a model for the preovulatory LH surge. A Y1 receptor antagonist (BIBO3304) was infused (25 microg/h) into the third cerebral ventricle (III-V) from 2 h before EB injection for 24 h, and had no effect on the ensuing LH surge. Using in situ hybridization, we then examined expression of NPY mRNA in the arcuate nucleus during the luteal, follicular and oestrous phases of the oestrous cycle, and found that levels were greatest during the luteal phase. Thus, reduced NPY synthesis might be an integral factor in the events leading to the cyclic preovulatory LH surge. This was tested by infusion of NPY (25 microg/h) into the III-V (as above). The NPY infusion delayed the LH surge until the infusion was ceased. High levels of NPY expression during the luteal phase of the oestrous cycle may be caused by progesterone. Thus, we determined whether NPY cells possess progesterone receptors (PR) and whether progesterone treatment up-regulates NPY mRNA expression in the arcuate nucleus. Immunohistochemistry for NPY and PR was performed in OVX, oestrogen-treated ewes, but no NPY cells of the arcuate nucleus were seen to colocalize PR. In situ hybridization for NPY was performed in OVX and OVX ewes treated with progesterone. There was no significant effect of progesterone treatment on NPY mRNA expression in the arcuate nucleus. We conclude that chronically elevated levels of NPY block the preovulatory surge of gonadotropin-releasing hormone/LH secretion in sheep, but high levels of NPY mRNA expression in the luteal phase of the oestrous cycle cannot be explained by an action of progesterone.

The retrograde tracer, FluoroGold, was used to trace the neuronal inputs from the septum, hypothalamus, and brain stem to the region of the GnRH neurons in the rostral preoptic area of the ram and to compare these imputs with those in the ewe. Sex differences were found in the number of retrogradely labeled cells in the dorsomedial and ventromedial nuclei. Retrogradely labeled cells were also observed in the lateral septum, preoptic area, organum vasculosum of the lamina terminalis, bed nucleus of the stria terminalis, stria terminalis, subfornical organ, periventricular nucleus, anterior hypothalamic area, lateral hypothalamus, arcuate nucleus, and posterior hypothalamus. These sex differences may partially explain sex differences in how GnRH secretion is regulated. Fluorescence immunohistochemistry was used to determine the neurochemical identity of some of these cells in the ram. Very few tyrosine hydroxylase-containing neurons in the A14 group (

There is considerable interest in the potential for measuring cortisol in hair as a means of quantifying stress responses in human and non-human animals. This review updates the rapid advancement in our knowledge of hair cortisol, methods for its measurement, its relationship to acute and chronic stress, and its repeatability and heritability. The advantages of measuring cortisol in hair compared with other matrices such as blood, saliva and excreta and the current theories of the mechanisms of cortisol incorporation into the fibre are described. Hair cortisol as a measure of the physiological response to stress in a variety of species is presented, including correlations with other sample matrices, the relationship between hair cortisol and psychosocial stress and the repeatability and heritability of hair cortisol concentrations. Current standards for the quantification of hair cortisol are critically reviewed in detail for the first time and gaps in technical validation of these methods highlighted. The known effects of a variety of sources of hair cortisol variation are also reviewed, including hair sampling site, sex, age and adiposity. There is currently insufficient evidence to conclude that cortisol concentration in hair accurately reflects long-term blood cortisol concentrations. Similarly, there is a lack of information surrounding the mechanisms of cortisol incorporation into the hair. This review highlights several directions for future research to more fully validate the use of hair cortisol as an indicator of chronic stress.

Increased adiposity is often associated with over activation of the hypothalamo-pituitary adrenal axis (HPA axis) and the sympatho-adrenal medullary system (SAM system) and excessive activation of these pathways in response to physiological challenges may be linked with the development of diseases. We tested the hypothesis that overweight/obese men aged 50–70 years will have greater HPA axis and SAM system responses to food intake compared with age matched lean men. Lean (Body Mass Index; BMI = 20-25 kg/m2; n = 19) and overweight/obese (BMI = 27-35 kg/m2; n = 17) men (50–70 years) made their own lunch using standardised ingredients at 1200 h. Concentrations of cortisol and alpha amylase were measured in saliva samples collected every 15 min from 1145 h-1400 h with the exception of during lunch (1215 h) where no sample was collected. Blood pressures and heart rate were measured at 1145 h and every 15 minutes between 1245 h and 1400 h. Overweight/obese men had significantly higher body weight, BMI, percentage body fat and waist and hip circumferences compared to lean men (p

Poor reproductive performance of Merino ewe flocks when mated to Border Leicester rams during spring may be due to seasonality of the Border Leicester breed. Two approaches were taken to test this assumption. Six young (12 months old) or six mixed-age (12, 24 and ≥36 months old) Border Leicester rams were either treated or not treated with melatonin implants (2 × 2 design) 6 weeks before the four groups of rams were each put with approximately 300 Merino ewes for an 8-week mating period. Implants were inserted in early September (experiment 1). The second approach was to yard or not yard ewes and mixed-age rams on several occasions during the first 3 weeks of the mating period (experiment 2). Pregnancy rate and twinning percentage were assessed by ultrasonography. In experiment 1, melatonin treatment in young rams increased (p0.001) pregnancy rate from 5.0% to 92.6%, but mixed-age rams did not respond (90.7% vs 89.5% for melatonin and non-melatonin treatments, respectively). Twinning rate was similar (p0.05) for ewes mated to either melatonin or non-melatonin-treated young rams (36.8% vs 40.0%, respectively), whereas melatonin significantly improved (p0.05) twinning rate in those ewes mated to mixed-age rams (49.1% vs 36.1%). After 6 weeks of melatonin treatment, scrotal circumference was greater (p0.05) in both young and mixed-aged rams than in untreated counterparts. In experiment 2, yarding of ewes and rams overnight on several occasions early in the mating period reduced (p0.001) pregnancy rate compared with non-yarded counterparts (89.5% vs 65.5%). Twinning rate was not affected (37.7% vs 36.1%, respectively). In summary, melatonin treatment of Border Leicester rams significantly improved flock reproductive performance in spring due to improved pregnancy rates with young rams and improved litter size with mixed-age rams.

Changes in the secretion of GH induced by long-term alterations in nutritional status are thought to result from alterations in somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) at the level of the hypothalamus. To date however, the effect of nutrition on the gene expression of SRIF and GHRH in a species where GH secretion is increased by food restriction, as is the case for the sheep and human, remains unknown. We determined the effect of under-nutrition on the expression of SRIF and GHRH in the hypothalamus of sheep. Ovariectomised ewes were randomly divided into two groups and either fed an ad lib diet (n=6) or a restricted diet of 500 g lucerne chaff per day (food-restricted; n=5) for 7 months. In situ hybridisation was used to study hypothalamic gene expression for GHRH, SRIF and galanin (GAL). The food-restricted animals had elevated plasma concentrations of GH; this was associated with an increase in GHRH mRNA levels in the arcuate nucleus (ARC) and reduced SRIF in the rostral periventricular nucleus and ventromedial hypothalamic nucleus. The level of gene expression of GAL in the ARC and SRIF in the caudal periventricular nucleus was similar in ad lib and food-restricted animals. In conclusion, the effect of chronic food-restriction on the secretion of GH reflects increased GHRH and reduced SRIF synthesis in the hypothalamus.

We measured the levels of progesterone receptor (PR) mRNA in the hypothalamus and preoptic area (POA) of the ewe across the oestrous cycle. Perfusion-fixed hypothalamic tissue was collected from sheep killed during the luteal and follicular phases and during behavioural oestrus. Blood samples taken at the time of tissue collection verified that the oestrous ewes were undergoing a preovulatory luteinizing hormone (LH) surge. Matched sections were taken from the POA, periventricular nucleus (PeVN), ventromedial nucleus (VMN) and arcuate nucleus of each animal. In situ hybridization was performed using a sheep specific, (35)S-labelled riboprobe for PR and semiquantitative image analysis was conducted on emulsion-dipped slides. The number of silver grains per cell was greater in the VMN and arcuate nucleus of oestrous ewes than in luteal and follicular phase ewes; there was no cyclic variation in the level of PR mRNA expression in the cells of the POA and PeVN. The number of labelled cells per mm2 in the VMN was higher in the oestrous ewes than in luteal phase and follicular phase ewes. The number of labelled cells in the PeVN was also higher in the oestrous ewes than in follicular phase ewes, but there was no cyclic variation in the POA and arcuate nucleus. In the ewe, the onset of behavioural oestrus corresponds to the onset of the preovulatory LH surge and increased PR mRNA expression at this time is likely to be due to the high concentrations of circulating oestrogen that precede this period.

This experiment determined if the degree of stimulation of the pituitary gland by GnRH affects the suppressive actions of inhibin and testosterone on gonadotropin secretion in rams. Two groups (n = 5) of castrated adult rams underwent hypothalamopituitary disconnection and were given two i.v. injections of vehicle or 0.64 microg/kg of recombinant human inhibin A (rh-inhibin) 6 h apart when treated with i.m. injections of oil and testosterone propionate every 12 h for at least 7 days. Each treatment was administered when the rams were infused i.v. with 125 ng of GnRH every 4 h (i.e., slow-pulse frequency) and 125 ng of GnRH every hour (i.e., fast-pulse frequency). The FSH concentrations and LH pulse amplitude were lower and the LH concentrations higher during the fast GnRH pulse frequency. The GnRH pulse frequency did not influence the ability of rh-inhibin and testosterone to suppress FSH secretion. Testosterone did not affect LH secretion. Following rh-inhibin treatment, LH pulse amplitude decreased at the slow, but not at the fast, GnRH pulse frequency, and LH concentrations decreased at both GnRH pulse frequencies. We conclude that the degree of stimulation of the pituitary by GnRH does not influence the ability of inhibin or testosterone to suppress FSH secretion in rams. Inhibin may be capable of suppressing LH secretion under conditions of low GnRH.

The sites of action and mechanisms by which gonadal steroids regulate gonadotrophin-releasing hormone (GnRH) in domestic animals remain largely unknown. This review summarises information gained from sheep regarding the distribution of the gonadal steroid receptors in the brain, the neurochemical identity and the projections of these steroid receptor-containing neurones. The cells in the hypothalamus that contain each of the gonadal steroid receptors (oestrogen receptor alpha (ERalpha), oestrogen receptor beta (ERbeta), progesterone receptor (PR) and androgen receptor (AR)) show a remarkably similar distribution, although the PR and AR-containing cells are less widespread than oestrogen receptors (ERs). There is considerable overlap in the distribution of ERalpha- and ERbeta-containing cells but also some unique sites for each subtype. This suggests differential regulation of the actions of oestrogen. There appears to be little sexual dimorphism in the distribution of the gonadal steroid receptors in the hypothalamus, with the notable exception of the ventromedial nucleus where females appear to have greater numbers of both ERalpha- and ERbeta-containing cells. Neuronal tracing studies have identified projections of some of the ERalpha-containing cells to sites that may allow interaction with the GnRH system. The receptor mapping, neuronal tracing and microimplantation studies suggest that the ventromedial nucleus is likely to be a key hypothalamic nucleus in the steroid regulation of GnRH secretion in sheep.

We have developed a ruminant model to study long-term alterations in adiposity on the expression of appetite-regulating peptides in the hypothalamus. In this model endocrine and metabolic status are fully defined as well as body composition. The current study sought to define the effects of altered adiposity on the expression of genes for neuropeptide Y (NPY), POMC, enkephalin (ENK), and melanin-concentrating hormone (MCH). Ovariectomized ewes with high (60 ± 1 kg) (FAT) or low (37 ± 3 kg) body weights (THIN) were blood sampled every 10 min for 8 h to determine metabolic and endocrine status. The animals were then killed and the brains perfused for in situ hybridization. Body composition analysis was performed on the carcass using dual energy x-ray absorptiometry; this indicated that the FAT animals were 36 ± 1% fat, whereas the THIN animals were 15 ± 2% fat. The LH interpulse interval was lower and mean GH concentrations were higher in the THIN animals; cortisol and TSH levels were not different between ...

We tested the hypothesis that sustained and repeated acute elevation of cortisol would impair the LH surge, estrus, and ovulation in gilts. Cortisol was injected intramuscularly, to achieve a sustained elevation of plasma concentrations of cortisol, or intravenously, to achieve an acute elevation of plasma concentrations of cortisol. Control gilts received i.m. injections of oil and i.v. injections of saline. These treatments were administered to gilts (n = 6 per treatment) at 12-h intervals from Days 7 to 11 of the estrous cycle until after estrus ceased or until Day 27 or 28 of the estrous cycle, whichever came first. The repeated acute elevation of cortisol had no effect on the LH surge, estrus, or ovulation. In contrast, when the elevation of cortisol was sustained, the LH surge, estrus, and ovulation were inhibited. We conclude that cortisol is capable of direct actions to impair reproductive processes in female pigs but that plasma concentrations of cortisol need to be elevated for a substantial period for this to occur.

This study tested the hypothesis that central administration of corticotrophin-releasing hormone (CRH) and/or arginine vasopressin (AVP) will affect the secretion of LH in rams and that testosterone is necessary for these actions to occur. Plasma LH levels were measured in castrated rams during 1 h infusion of either 100 microliter vehicle/mock cerebrospinal fluid (CSF) or mock CSF containing 25 microgram CRH, 25 microgram AVP or 25 microgram of each peptide through guide cannulae into the third cerebral ventricle. These intracerebroventricular (i.c.v.) infusions were given to the castrated rams following injections (i.m.) each 12 h of oil or 8 mg testosterone propionate for 7 days. Blood samples were collected every 10 min for 4 h before i.c.v. infusion, during infusion and for 4 h following the infusion. Infusion of vehicle did not affect any endocrine parameters. In contrast, the plasma concentrations of LH and the amplitude of LH pulses were increased significantly during and following infusion of CRH, and this effect was not influenced by whether the castrated rams were treated with testosterone propionate or whether the CRH was administered in combination with AVP. Infusion of AVP alone did not affect LH secretion. The frequency of LH pulses and the plasma concentrations of FSH did not change with any of the i.c.v. treatments. The plasma concentrations of cortisol were significantly increased by CRH and AVP infusions. The plasma concentrations of cortisol achieved during and following i.c.v. infusion of CRH and AVP combined were greater than the concentrations achieved as a result of treatment with AVP alone but were similar to those with CRH. There was no effect of testosterone propionate on cortisol levels. These results show that CRH, but not AVP, is capable of acting either centrally or at the pituitary level to increase the secretion of LH in rams and these actions are not affected by testosterone. The stimulatory effects of CRH on LH secretion are to increase the amplitude of GnRH pulses and/or the responsiveness of the pituitary to the actions of GnRH with no effect on the frequency of GnRH pulses. The secretion of FSH in rams is not influenced by either CRH or AVP. The effect of CRH to increase LH pulse amplitude occurs in the face of increased cortisol levels, further reinforcing our belief that this adrenal steroid does not affect the reproductive axis in this species.

Three experiments were conducted with castrated Romney Marsh rams (wethers) to investigate the ability of testosterone and inhibin to suppress the secretion of LH and FSH during the breeding and the non-breeding seasons. In Experiment 1, wethers (n=5/group) were treated every 12 h for 7 days with oil or 16 mg testosterone propionate (i.m.) and were then given two i.v. injections either of vehicle or of 0.64 microg/kg human recombinant inhibin A (hr-inhibin) 6 h apart. Blood samples were collected for 4 h before inhibin or vehicle treatment and for 6 h afterwards for the assay of LH and FSH. In Experiments 2 and 3 wethers underwent hypothalamo-pituitary disconnection (HPD) and were given 125 ng GnRH i.v. every 2 h. In Experiment 2, HPD wethers (n=3/group) were injected (i.m.) every 12 h with oil or testosterone and blood samples were collected over 9 h before treatment and 7 days after treatment. In Experiment 3, HPD (n=5/group) wethers were treated with vehicle or hr-inhibin, as in Experiment 1, after treatment with oil, or 4, 8 or 16 mg testosterone twice daily for 7 days. Blood samples were collected over 4 h before treatment with vehicle or hr-inhibin and for 6 h afterwards. Treatment of wethers with testosterone (Experiment 1) resulted in a significant decrease in the plasma concentrations of LH and number of LH pulses per hour but the magnitude of these reductions did not differ between seasons. Testosterone treatment had no effect on LH secretion in GnRH-pulsed HPD wethers in either season and treatment with hr-inhibin did not affect LH secretion in wethers or HPD wethers in any instance. Plasma concentrations of FSH were significantly (P

1. Angiotensin II (AngII) is well recognized as a regulator of adrenal cortisol production in the sheep in vivo, but studies to date have failed to reveal this action on cultured ovine adrenocortical cells. Similarly, phorbol myristate acetate (PMA), an activator of protein kinase C, also has little effect on cortisol production from ovine adrenocortical cells in vitro. Previous studies have, however, only looked at the responses to single concentrations of AngII or PMA, but have led to the suggestion that important differences exist between adrenocortical cells of the sheep and other species. 2. We have extended previous studies by examining cortisol production by ovine adrenocortical cells over 3 h in response to a broad range of concentrations of either AngII (10(-10) to 10(-6) mol/L) or PMA (10(-10) to 10(-5) mol/L). In addition, we have investigated the possible role of gonadal factors in regulating these responses by comparing cultures derived from intact and gonadectomized male and female sheep. 3. Angiotensin II caused concentration-dependent increases (P < 0.05) in cortisol production in all cultures, with cortisol production in response to AngII being greater (P < 0.05) in cultures from intact male sheep than in the cultures from orchidectomized males or intact females. Cortisol production in response to AngII was modest in all groups (two- to 2.6-fold increases). 4. Phorbol myristate acetate elicited a concentration-dependent inhibition (P < 0.01) of basal cortisol production in adrenocortical cultures from all groups. Phorbol myristate acetate produced a greater inhibition (P < 0.05) of cortisol production in cultures from intact male sheep than either the orchidectomized males or intact females. 5. These findings demonstrate that ovine adrenocortical cells are responsive in a concentration-dependent fashion to AngII and PMA. In addition, sex and gonadal factors may play an important role in regulating the responsiveness of adrenocortical cells to these factors.

We investigated the effects of repeated acute activation of the hypothalamo-pituitary adrenal axis, prior to and during estrus, on reproduction in gilts. Individual gilts (n = 24 per treatment) either served as controls or were subjected to daily acute stress ("negative handling," brief electric shock with a battery-operated prodder during confinement with the experimenter) commencing, on average, 8 days prior to estrus. Gilts subjected to negative handling had a significant elevation in plasma concentrations of cortisol that lasted at least 3-4 h, and these gilts were slower than control gilts to approach and interact with the experimenter in a standard test. Nevertheless, reproductive performance--as measured by sexual receptivity and proceptivity, ovulation, the percentage of gilts that became pregnant, the number of embryos 20-21 days after insemination, and the weight of embryos--was not affected by repeated acute activation of the hypothalamo-pituitary adrenal axis. Our results suggest that repeated acute activation of the hypothalamo-pituitary adrenal axis prior to and during estrus does not affect the factors that control estrus and ovulation in gilts.

It has been proposed that short-term activation of the hypothalamo-pituitary adrenal axis, with a consequent increase in the secretion of cortisol, amy disrupt the endocrine events prior to ovulation and thereby impair reproduction in females. We investigated this concept in gilts in which oestrus was detected by introduction to boars, where intense physical contact is possible, or by applying pressure to the back of gilts (back-pressure test) during fence-line exposure to boars, where intense physical contact is prohibited. We expected that there would be a greater release of cortisol and that reproduction would be inhibited in gilts introduced to boars compared to gilts in which the back-pressure test was used. As expected, introduction of gilts to boars resulted in a significant transient increase in plasma concentrations of cortisol while there was no significant effect of using the back-pressure test on plasma cortisol. Nevertheless, introduction of gilts to boars did not impair reproduction and there was no effect of method of detecting oestrus on duration of oestrus, sexual receptivity, fertility or fecundity. The length of the oestrous cycle was decreased and ovulation rate increased in gilts that were introduced to boars compared to gilts that underwent the back-pressure test, indicating that introduction of gilts to boars may have stimulated these aspects of reproduction. These stimulatory effects may have been due to an increased exposure of gilts to sexual behaviour and stimuli from boars when introduced to boars and/or to stimulatory effects of the hypothalamo-pituitary adrenal axis on some aspects of reproduction.

We have tested the hypothesis that treatment of castrated rams (wethers) with human recombinant follistatin-288 (FS-288) suppresses plasma concentrations of FSH but has no effect on plasma concentrations of LH. Wethers were given an i.v. injection of vehicle or 50 micrograms FS-288 followed by a 12-h i.v. infusion of vehicle or 800 micrograms FS-288, respectively. This dose and treatment regimen was identical to that used in a previous study in which wethers were treated with vehicle or human recombinant inhibin A (hr-inhibin). Human recombinant follistatin significantly (p < 0.05) suppressed plasma concentrations of FSH, but vehicle had no effect. The maximal suppression of FSH occurred 12-15 h after the start of treatment, at which time the plasma concentrations were reduced by 20.6%. The reduction in plasma concentrations of FSH caused by FS-288 was about 2.6-fold less than that observed after the same treatment with hr-inhibin. FS-288 had no effect on plasma concentrations of LH; this result was similar to the findings we obtained with hr-inhibin. These data suggest that follistatin is a less potent negative regulator of FSH secretion than inhibin and that follistatin is not a significant regulator of the secretion of LH in rams.

We investigated the effect of genetic selection for temperament on the way that stressors affect the behaviour and the adrenal and reproductive axes of sheep. We tested three hypotheses: (i) isolation would increase cortisol secretion and decrease luteinising hormone (LH) secretion more in nervous sheep than in calm sheep; (ii) isolation combined with simulated human presence would increase cortisol secretion and decrease LH secretion more in nervous sheep than in calm sheep and (iii) isolation combined with stressors that were not specific to the selection process (i.e. non-selection stressors) would increase cortisol secretion and decrease LH secretion equally in calm and nervous sheep. Isolation alone increased cortisol secretion and decreased LH secretion in nervous sheep but not in calm sheep. Compared to calm sheep, nervous sheep were more agitated during the first 2 h of isolation but not during the second 2 h of isolation. Exposure to non-selection stressors increased cortisol secretion, decreased LH pulse amplitude and the mean plasma concentrations of LH in both calm and nervous sheep. We conclude that genetic selection for temperament affects the behavioural expression of the stress response and the secretion of adrenal and reproductive hormones during isolation, but has less impact on their reactivity to non-selection stressors.